DeepCell Kiosk: scaling deep learning-enabled cellular image analysis with Kubernetes.

Deep learning is transforming the analysis of biological images, but applying these models to large datasets remains challenging. Here we describe the DeepCell Kiosk, cloud-native software that dynamically scales deep learning workflows to accommodate large imaging datasets. To demonstrate the scalability and affordability of this software, we identified cell nuclei in 106 1-megapixel images in ~5.5 h for ~US$250, with a cost below US$100 achievable depending on cluster configuration. The DeepCell Kiosk can be downloaded at https://github.com/vanvalenlab/kiosk-console ; a persistent deployment is available at https://deepcell.org/ .

The robotic-assisted free jejunal flap for neovaginal canal creation in gender-affirming vaginoplasties.

INTRODUCTION: One of the biggest challenges with gender-affirming vaginoplasty was the creation of a long-lasting, durable, patent, and self-lubricating neovaginal canal that allowed for spontaneous, pain-free sexual intercourse. The jejunum was a durable, physiologic, and intestinal option to create the neovaginal canal that minimizes the adverse effects of skin graft, peritoneal, and colonic vaginoplasties. Free jejunal vaginoplasties had been performed in cis females for congenital genitourinary anomalies like Mullerian agenesis or after gynecologic-oncologic surgery but had yet to be reported for gender-affirming vaginoplasties. The purpose of this report was to present a technique for a physiologic, intestinal, gender-affirming vaginoplasty without the disadvantages of colonic vaginoplasties. PATIENTS AND METHODS: This report presented six patients, all natal males who identified as female, undergoing robotic-assisted free jejunal flap gender-affirming vaginoplasty. Mean age was 35.8 years (range: 21-66). Mean body mass index was 33.2 kg/m2 (range: 28.0-41.0). The proximal aspect of the neovaginal canal was created intra-abdominally by elevating peritoneal flaps from the posterior bladder wall to be reflected downward into the external neovaginal canal. The jejunal flap was harvested. The greater saphenous vein was harvested to create an arteriovenous loop between the flap vessels and the recipient femoral artery in an end-to-side fashion and a branch of the femoral vein. The jejunal flap was passed intra-abdominally through the groin incision and then trans-peritoneally into the neovaginal canal. The jejunal segment was inset to the proximal peritoneal flaps and the distal inverted penoscrotal skin of the neovaginal introitus. RESULTS: Mean length of the harvest jejunal segment was 19.2 cm (range: 15-20). Mean time to ambulation, foley removal, and first vaginal dilation were 3.3 (range: 3-4), 4.0 (range: 3-5), and 4.5 days (range: 4-6), respectively. By a mean follow-up duration of 8.0 months (range: 1-14), mean vaginal depth and diameter were 7.0 and 1.3 cm (range: 1.0-1.5), respectively. Two (33.3%) patients experienced postoperative complications, including groin hematoma (n = 1, 16.7%) and reoperation for correction of dehiscence of the jejunal flap to the vaginal introitus (n = 1, 16.7%). CONCLUSION: Gender-affirming surgeons should consider a free vascularized segment of jejunum as an option to line the neovaginal canal in the correct patients.

Primary Targeted Muscle Reinnervation in Above-Knee Amputations in Patients with Unsalvageable Limbs from Limb-Threatening Ischemia or Infection.

BACKGROUND: Amputees frequently suffer from chronic pain in both their residual limbs (RLP) and phantom limbs (PLP) following their amputation. Targeted muscle reinnervation (TMR) is a nerve transfer technique that has been demonstrated to improve pain secondarily and at time of amputation. The goal of this study is to report on the efficacy of primary TMR at time of above-knee level amputations in the setting of limb-threatening ischemia or infection. METHODS: This is a retrospective review of a single-surgeon experience with TMR in patients undergoing through- or above-knee level amputations from January 2018 to June 2021. Patient charts were reviewed for the comorbidities in the Charlson Comorbidity Index. Postoperative notes were assayed for presence and absence of RLP and PLP, overall pain severity, chronic narcotic use, ambulatory status, and complications. A control group of patients undergoing lower limb amputation who did not receive TMR from January 2014 to December 2017 was used for comparison. RESULTS: Forty-one patients with through- or above-knee level amputations and primary TMR were included in this study. The tibial and common peroneal nerves were transferred in all cases to motor branches to the gastrocnemius, semimembranosus, semitendinosus, and biceps femoris. Fifty-eight patients with through- or above-knee level amputations without TMR were included for comparison. The TMR group had significantly less overall pain (41.5 vs. 67.2%, p = 0.01), RLP (26.8 vs. 44.8%, p = 0.04), and PLP (19.5 vs. 43.1%, p = 0.02). There were no significant differences in complication rates. CONCLUSION: TMR can safely and effectively be performed at time of a through- and above-knee level amputation and improves pain outcomes.

Health-related quality of life and pain outcomes with [177Lu]Lu-PSMA-617 plus standard of care versus standard of care in patients with metastatic castration-resistant prostate cancer (VISION): a multicentre, open-label, randomised, phase 3 trial.

BACKGROUND: In VISION, the prostate-specific membrane antigen (PSMA)-targeted radioligand therapy lutetium-177 [177Lu]Lu-PSMA-617 (vipivotide tetraxetan) improved radiographic progression-free survival and overall survival when added to protocol-permitted standard of care in patients with metastatic castration-resistant prostate cancer. Here, we report additional health-related quality of life (HRQOL), pain, and symptomatic skeletal event results. METHODS: This multicentre, open-label, randomised, phase 3 trial was conducted at 84 cancer centres in nine countries in North America and Europe. Eligible patients were aged 18 years or older; had progressive PSMA-positive metastatic castration-resistant prostate cancer; an Eastern Cooperative Oncology Group (ECOG) performance status score of 0-2; and had previously received of at least one androgen receptor pathway inhibitor and one or two taxane-containing regimens. Patients were randomly assigned (2:1) to receive either [177Lu]Lu-PSMA-617 plus protocol-permitted standard of care ([177Lu]Lu-PSMA-617 group) or standard of care alone (control group) using permuted blocks. Randomisation was stratified by baseline lactate dehydrogenase concentration, liver metastases, ECOG performance status, and androgen receptor pathway inhibitor inclusion in standard of care. Patients in the [177Lu]Lu-PSMA-617 group received intravenous infusions of 7·4 gigabecquerel (GBq; 200 millicurie [mCi]) [177Lu]Lu-PSMA-617 every 6 weeks for four cycles plus two optional additional cycles. Standard of care included approved hormonal treatments, bisphosphonates, and radiotherapy. The alternate primary endpoints were radiographic progression-free survival and overall survival, which have been reported. Here we report the key secondary endpoint of time to first symptomatic skeletal event, and other secondary endpoints of HRQOL assessed with the Functional Assessment of Cancer Therapy-Prostate (FACT-P) and EQ-5D-5L, and pain assessed with the Brief Pain Inventory-Short Form (BPI-SF). Patient-reported outcomes and symptomatic skeletal events were analysed in all patients who were randomly assigned after implementation of measures designed to reduce the dropout rate in the control group (on or after March 5, 2019), and safety was analysed according to treatment received in all patients who received at least one dose of treatment. This trial is registered with ClinicalTrials.gov, NCT03511664, and is active but not recruiting. FINDINGS: Between June 4, 2018, and Oct 23, 2019, 831 patients were enrolled, of whom 581 were randomly assigned to the [177Lu]Lu-PSMA-617 group (n=385) or control group (n=196) on or after March 5, 2019, and were included in analyses of HRQOL, pain, and time to first symptomatic skeletal event. The median age of patients was 71 years (IQR 65-75) in the [177Lu]Lu-PSMA-617 group and 72·0 years (66-76) in the control group. Median time to first symptomatic skeletal event or death was 11·5 months (95% CI 10·3-13·2) in the [177Lu]Lu-PSMA-617 group and 6·8 months (5·2-8·5) in the control group (hazard ratio [HR] 0·50, 95% CI 0·40-0·62). Time to worsening was delayed in the [177Lu]Lu-PSMA-617 group versus the control group for FACT-P score (HR 0·54, 0·45-0·66) and subdomains, BPI-SF pain intensity score (0·52, 0·42-0·63), and EQ-5D-5L utility score (0·65, 0·54-0·78). Grade 3 or 4 haematological adverse events included decreased haemoglobin (80 [15%] of 529 assessable patients who received [177Lu]Lu-PSMA-617 plus standard of care vs 13 [6%] of 205 who received standard of care only), lymphocyte concentrations (269 [51%] vs 39 [19%]), and platelet counts (49 [9%] vs five [2%]). Treatment-related adverse events leading to death occurred in five (1%) patients who received [177Lu]Lu-PSMA-617 plus standard of care (pancytopenia [n=2], bone marrow failure [n=1], subdural haematoma [n=1], and intracranial haemorrhage [n=1]) and no patients who received standard of care only. INTERPRETATION: [177Lu]Lu-PSMA-617 plus standard of care delayed time to worsening in HRQOL and time to skeletal events compared with standard of care alone. These findings support the use of [177Lu]Lu-PSMA-617 in patients with metastatic castration-resistant prostate cancer who received previous androgen receptor pathway inhibitor and taxane treatment. FUNDING: Advanced Accelerator Applications (Novartis).

Nephrocalcinosis in children who received high-dose vitamin D.

BACKGROUND: Vitamin D supplements are readily available as over-the-counter preparations. However, although rare, cases of vitamin D overdose still occur and are associated with nephrocalcinosis and life-threatening hypercalcemia. Errors in manufacturing of nutritional supplements may be a cause of vitamin D intoxication in children. This study aimed to identify factors associated with vitamin D overdose-related nephrocalcinosis in children due to manufacturing errors in supplements. METHODS: This retrospective study reviewed medical charts of pediatric patients with non-registered supplement-related vitamin D overdose at a tertiary referral hospital between 2006 and 2011. Clinical and laboratory characteristics of patients with or without nephrocalcinosis were evaluated. Receiver operating characteristics curve and area under the receiver operating characteristics curve were used to determine the most predictive value of each characteristic. RESULTS: A total of 44 patients (males: 29; age: 7-62 months) were included. Age ≤ 16.5 months, body weight ≤ 10.25 kg, body height ≤ 78.5 cm, body surface area (BSA) ≤ 0.475 m2, 25-hydroxyvitamin D3 ≥ 143 ng/mL, and calcium ≥ 10.65 mg/dL were predictive of developing nephrocalcinosis with a sensitivity and specificity of > 60%. Univariant analysis revealed that BSA was the most significant anthropometric prognostic factor (odds ratio: 12.09; 95% confidence interval: 2.61-55.72; P = 0.001). CONCLUSIONS: Children with smaller BSAs were more vulnerable to high-dose vitamin D3-related nephrocalcinosis. Physicians and parents should be aware of the potential adverse effects of vitamin D overdose in children. A higher resolution version of the Graphical abstract is available as Supplementary information.

Analysis of urgent/emergent conversions from monitored anesthesia care to general anesthesia with airway instrumentation.

BACKGROUND: Monitored Anesthesia Care (MAC) is an anesthetic service involving the titration of sedatives/analgesics to achieve varying levels of sedation while avoiding general anesthesia (GA) and airway instrumentation. The goal of our study was to determine the overall incidence of conversion from MAC to general anesthesia with airway instrumentation and elucidate reasons and risk factors for conversion. METHODS: In this retrospective observational study, all non-obstetric adult patients who received MAC from July 2002 to July 2015 at Mount Sinai Hospital were electronically screened for inclusion via a clinical database. Patient, procedure, anesthetic, and practitioner data were all collected and analyzed to generate descriptive analyses. Subsequent univariate and multivariate analyses were used to identify specific risk factors associated with conversion to GA. RESULTS: Overall, 0.50% (1097/219,061) of MAC cases were converted to GA. Approximately half of conversions were due to the patient's "intolerance" of MAC (with or without failed regional anesthesia), while the other half were due to physiologic derangements. Body mass index, male sex, American Society of Anesthesiologists Physical Status Classification, anesthesia team composition, and surgical specialty were all associated with risk of conversion to GA. CONCLUSIONS: This is one of the first and largest retrospective studies aimed at identifying reasons and risk factors associated with the conversion of MAC to GA. These findings may be used to help better anticipate or prevent these events.

Impact of concomitant vasoactive treatment and mechanical left ventricular unloading in a porcine model of profound cardiogenic shock.

BACKGROUND: Concomitant vasoactive drugs are often required to maintain adequate perfusion pressure in patients with acute myocardial infarction (AMI) and cardiogenic shock (CS) receiving hemodynamic support with an axial flow pump (Impella CP). OBJECTIVE: To compare the effect of equipotent dosages of epinephrine, dopamine, norepinephrine, and phenylephrine on cardiac work and end-organ perfusion in a porcine model of profound ischemic CS supported with an Impella CP. METHODS: CS was induced in 10 pigs by stepwise intracoronary injection of polyvinyl microspheres. Hemodynamic support with Impella CP was initiated followed by blinded crossover to vasoactive treatment with norepinephrine (0.10 µg/kg/min), epinephrine (0.10 µg/kg/min), or dopamine (10 µg/kg/min) for 30 min each. At the end of the study, phenylephrine (10 µg/kg/min) was administered for 20 min. The primary outcome was cardiac workload, a product of pressure-volume area (PVA) and heart rate (HR), measured using the conductance catheter technique. End-organ perfusion was assessed by measuring venous oxygen saturation from the pulmonary artery (SvO2), jugular bulb, and renal vein. Treatment effects were evaluated using multilevel mixed-effects linear regression. RESULTS: All catecholamines significantly increased LV stroke work and cardiac work, dopamine to the greatest extend by 341.8 × 103 (mmHg × mL)/min [95% CI (174.1, 509.5), p < 0.0001], and SvO2 significantly improved during all catecholamines. Phenylephrine, a vasoconstrictor, caused a significant increase in cardiac work by 437.8 × 103 (mmHg × mL)/min [95% CI (297.9, 577.6), p < 0.0001] due to increase in potential energy (p = 0.001), but no significant change in LV stroke work. Also, phenylephrine tended to decrease SvO2 (p = 0.063) and increased arterial lactate levels (p = 0.002). CONCLUSION: Catecholamines increased end-organ perfusion at the expense of increased cardiac work, most by dopamine. However, phenylephrine increased cardiac work with no increase in end-organ perfusion.

Defining Risk of Postoperative Obstructive Sleep Apnea in Patients With 22q11.2DS Undergoing Pharyngeal Flap Surgery for Velopharyngeal Dysfunction Using Polysomnographic Evaluation.

OBJECTIVE: To determine pre- and postoperative prevalence of obstructive sleep apnea (OSA) in patients with 22q11.2 deletion syndrome (DS) undergoing wide posterior pharyngeal flap (PPF) surgery for velopharyngeal dysfunction (VPD). DESIGN: Retrospective study using pre- and postoperative polysomnography (PSG) to determine prevalence of OSA. Medical records were reviewed for patients' medical comorbidities. Parents were surveyed about snoring. SETTING: Academic tertiary care pediatric hospital. PATIENTS: Forty patients with laboratory confirmed 22q11.2DS followed over a 6-year period. INTERVENTIONS: Pre- and postoperative PSG, speech evaluation, and parent surveys. MAIN OUTCOME MEASURE: Severity and prevalence of OSA, defined by obstructive apnea hypopnea index (OAHI), before and after PPF surgery to determine whether PPF is associated with increased risk of OSA. RESULTS: Mean OAHI did not change significantly after PPF surgery (1.1/h vs 2.1/h, P = .330). Prevalence of clinically significant OSA (OAHI ≥ 5) was identical pre- and postoperatively (2 of 40), with both cases having severe-range OSA requiring positive airway pressure therapy. All other patients had mild-range OSA. Nasal resonance was graded as severe preoperatively in 85% of patients. None were graded as severe postoperatively. No single patient factor or parent-reported concern predicted risk of OSA (OAHI ≥ 1.5). CONCLUSIONS: Patients with 22q11.2DS are medically complex and are at increased risk of OSA at baseline. Wide PPF surgery for severe VPD does not significantly increase risk of OSA. Careful perioperative planning is essential to optimize both speech and sleep outcomes.

Investigating Multiple-Reported Bacterial Sexually Transmitted Infection Hot Spot Counties in the United States: Ordered Spatial Logistic Regression.

PURPOSE: To identify and examine the correlates of multiple bacterial sexually transmitted infection (STI) hot spot counties in the United States. METHODS: We assembled and analyzed 5 years (2008-2012) of cross-sectional STI morbidity data to identify multiple bacterial STI (chlamydia, gonorrhea, and syphilis) hot spot counties using hot spot analysis. Then, we examined the association between the multi-STI hot spots and select multiyear (2008-2012) sociodemographic factors (data obtained from the American Community Survey) using ordered spatial logistic regression analyses. RESULTS: Of the 2935 counties, the results indicated that 85 counties were hot spots for all 3 STIs (3-STI hot spot counties), 177 were hot spots for 2 STIs (2-STI hot spot counties), and 145 were hot spots for only 1 STI (1-STI hot spot counties). Approximately 93% (79 of 85) of the counties determined to be 3-STI hot spots were found in 4 southern states--Mississippi (n = 25), Arkansas (n = 22), Louisiana (n = 19), and Alabama (n = 13). Counties determined to be 2 STI hot spots were found in 7 southern states--Arkansas, Louisiana, Mississippi, Alabama, Georgia, and North and South Carolina had at least ten 2-STI hot spot counties each. The multi-STI hot spot classes were significantly (P < 0.05) associated with percent black (non-Hispanic), percent Hispanics, percent American Indians, population density, male-female sex ratio, percent aged 25 to 44 years, and violent crime rate. CONCLUSIONS: This study provides information on multiple STI hot spot counties in the United States and the associated sociodemographic factors. Such information can be used to assist planning, designing, and implementing effective integrated bacterial STI prevention and control programs/interventions.

How seabirds plunge-dive without injuries.

In nature, several seabirds (e.g., gannets and boobies) dive into water at up to 24 m/s as a hunting mechanism; furthermore, gannets and boobies have a slender neck, which is potentially the weakest part of the body under compression during high-speed impact. In this work, we investigate the stability of the bird's neck during plunge-diving by understanding the interaction between the fluid forces acting on the head and the flexibility of the neck. First, we use a salvaged bird to identify plunge-diving phases. Anatomical features of the skull and neck were acquired to quantify the effect of beak geometry and neck musculature on the stability during a plunge-dive. Second, physical experiments using an elastic beam as a model for the neck attached to a skull-like cone revealed the limits for the stability of the neck during the bird's dive as a function of impact velocity and geometric factors. We find that the neck length, neck muscles, and diving speed of the bird predominantly reduce the likelihood of injury during the plunge-dive. Finally, we use our results to discuss maximum diving speeds for humans to avoid injury.

Does including violent crime rates in ecological regression models of sexually transmissible infection rates improve model quality? Insights from spatial regression analyses.

Background Violent crime rates are often correlated with the hard-to-measure social determinants of sexually transmissible infections (STIs). In this study, we examined whether including violent crime rate as an independent variable can improve the quality of ecological regression models of STIs. METHODS: We obtained multiyear (2008-12) cross-sectional county-level data on violent crime and three STIs (chlamydia, gonorrhoea, and primary and secondary (P&S) syphilis) from counties in all the contiguous states in the US (except Illinois and Florida, due to lack of data). We used two measures of STI morbidity (one categorical and one continuous) and applied spatial regression with the spatial error model for each STI, with and without violent crime rate as an independent variable. We computed the associated Akaike's information criterion (AIC) and Bayesian information criterion (BIC) as our measure of the relative goodness of fit of the models. RESULTS: Including the violent crime rate as an independent variable improved the quality of the regression models after controlling for several sociodemographic factors. We found that the lower calculated AICs and BICs indicated more favourable goodness of fit in all the models that included violent crime rates, except for the categorical P&S syphilis model, in which the violent crime variable was not statistically significant. CONCLUSION: Because violent crime rates can account for the hard-to-measure social determinants of STIs, including violent crime rate as an independent variable can improve ecological regression models of STIs.

Palatal Measurements Pre- and Post-Furlow Cleft Palate Repair: Analysis of Palatal Lengthening and Comparison Within Cleft Types.

OBJECTIVE: The purpose of this study was to characterize intraoperative palatal lengthening with the modified Furlow cleft palatal repair and to determine whether lengthening correlated with preoperative cleft width, cleft type, or operating surgeon. DESIGN: Retrospective study. SETTING: Academic tertiary care pediatric hospital. PATIENTS: One hundred eighty pediatric patients undergoing primary or secondary palatoplasty using the Furlow technique. INTERVENTIONS: Cleft and palatal lengths and widths were measured pre- and post-Furlow cleft palatal repair. MAIN OUTCOME MEASURES: Immediate postoperative percentage change in surface palate length, straight palate length, and soft palate length. RESULTS: The average cleft widest width and width at the hard-soft palate junction were 10.2 and 9.5 mm, respectively, and varied with Veau cleft type. Following Furlow palatoplasty, lengths of the curved, straight, and soft palate increased by 7.5%, 15.8%, and 30.6%, respectively. Degree of palatal lengthening varied among surgeons and Veau cleft type but was not related to cleft width. Seven (4.0%) patients developed postoperative oronasal fistulas. Patients with a Veau IV cleft and larger cleft widths were at an increased risk for fistula formation. CONCLUSIONS: This study demonstrates that overall palatal lengthening occurs with the modified Furlow technique. Long-term follow-up studies are needed to determine the clinical relevance of these findings.

Quantifying Pediatric Facial Palsy: : Using Digital Image Correlation to Objectively Characterize Pediatric Facial Symmetry.

OBJECTIVE: This study introduces digital image correlation (DIC) as a novel technology to objectively quantify pediatric facial symmetry. DESIGN: Descriptive cohort study of patients' facial symmetry as measured by DIC. SETTING: Academic tertiary care hospital. PATIENTS: 9 of 12 identified facial palsy and 13 of 26 identified control subjects participated. INTERVENTIONS: DIC was used to quantify facial strain and symmetry as patients made the 5 standard Sunnybrook facial expressions. Each subject was evaluated according to the Sunnybrook scale by 4 evaluators, 3 plastic surgeons, and 1 occupational therapist. MAIN OUTCOME MEASURE: The percentage asymmetry values were calculated and compared between the facial palsy and control groups using both DIC and Sunnybrook. RESULTS: Using DIC, facial palsy subjects had 32.99% asymmetry compared with 14.84% in controls (P < .01). Using Sunnybrook, facial palsy subjects had 24.11% asymmetry compared to 3.87% in controls (P < .01). The 2 metrics were positively correlated (P < .01). There was significant variability among the Sunnybrook evaluators (P = .02). CONCLUSIONS: DIC is a novel technique of objectively quantifying facial motion of the animated face. As surgical and medical approaches toward facial palsy expand, it is essential to have a means to compare results and improve patient outcomes.

Perioral Rejuvenation: A Prospective, Quantitative Dynamic Three-Dimensional Analysis of a Dual Modality Treatment.

BACKGROUND: The perioral region is the most dynamic anatomic area of the face and subject to complex and dramatic changes during aging. Successful treatment for perioral rejuvenation has yet to be identified, and prior studies have reported only subjective outcomes. OBJECTIVES: The purpose of this study was to utilize our validated dynamic 3-dimensional imaging technology to determine whether conservative neuromodulation combined with hyaluronic acid filler volumization can decrease perioral strain and increase volume with significant patient satisfaction. METHODS: An IRB-approved prospective study of a dual modality treatment for perioral rhytids was performed on females with perioral aging who had not had prior facial treatment within the past year. Eighteen (18) units of Dysport were injected into the upper and lower orbicularis oris and 1 cc of Restylane Silk was injected in volume-depleted perioral regions in each patient. Each patient underwent imaging with digital image correlation (DIC) and completed the FACE-Q survey prior to injection and at 14 days and 90 days postinjection. RESULTS: Thirty-two female patients were recruited. A significant reduction in perioral strain was observed at both day 14 and day 90. This was concomitant with a significant increase in perioral volume at day 14 that at 90 days was significantly retained in the marionette lines. Further, there was a significant improvement in patient satisfaction with overall facial appearance at day 14 that was maintained at 90 days. CONCLUSIONS: Conservative neuromodulation and hyaluronic acid filler volumization of the perioral region produces a significant reduction in strain correlating with high patient satisfaction, even at 90 days. This dual modality treatment is effective in rejuvenating the perioral region, and its future optimization will provide greater therapeutic options for this anatomically complex area.

Influence of Standardized Orientation on Patient Perception of Perioperative Care Following Alveolar Cleft Repair: A Survey Based Study of Patients Treated in a Large Academic Medical Center.

OBJECTIVE: This study aims to better understand patient-reported outcomes for iliac bone grafting surgery for alveolar cleft repair and to determine how standardizing perioperative patient instruction affects patient-reported outcomes. DESIGN: Retrospective survey-based assessment of patients undergoing iliac bone grafting with and without hospital-based systems standardization. SETTING: Academic tertiary care hospital. PATIENTS: Of the 195 identified patients, 127 participated. INTERVENTIONS: Survey on pain and satisfaction regarding iliac bone grafting surgery. MAIN OUTCOME MEASURES: Survey answers measured patient opinions about the surgery. Answers of the pre- and poststandardization patients were compared to determine the effect of standardizing patient instructions. RESULTS: Patients rated their satisfaction with the surgery and recovery a 4.5 and 4.4 out of 5, respectively. They rated their overall pain in the hospital a 5.5 out of 10 (4.9 in the mouth, 5.7 in the hip). Patients were discharged an average of 1.2 days after surgery and could return to normal daily activity in 6.1 days. Poststandardization patients were more likely to adhere to instructions regarding use of an antibacterial mouthrinse and a protective oral splint. CONCLUSIONS: Patients were highly satisfied with the iliac bone grafting procedure and the recovery and reported only moderate levels of postoperative pain. Implementing standardized patient instructions may not affect patient satisfaction or pain severity, but it significantly increased patient adherence to physician instructions.

Inactivation of the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath) by acetylene.

Acetylene (HCCH) has a long history as a mechanism-based enzyme inhibitor and is considered an active-site probe of the particulate methane monooxygenase (pMMO). Here, we report how HCCH inactivates pMMO in Methylococcus capsulatus (Bath) by using high-resolution mass spectrometry and computational simulation. High-resolution MALDI-TOF MS of intact pMMO complexes has allowed us to confirm that the enzyme oxidizes HCCH to the ketene (C2H2O) intermediate, which then forms an acetylation adduct with the transmembrane PmoC subunit. LC-MS/MS analysis of the peptides derived from in-gel proteolytic digestion of the protein subunit identifies K196 of PmoC as the site of acetylation. No evidence is obtained for chemical modification of the PmoA or PmoB subunit. The inactivation of pMMO by a single adduct in the transmembrane PmoC domain is intriguing given the complexity of the structural fold of this large membrane-protein complex as well as the complicated roles played by the various metal cofactors in the enzyme catalysis. Computational studies suggest that the entry of hydrophobic substrates to, and migration of products from, the catalytic site of pMMO are controlled tightly within the transmembrane domain. Support of these conclusions is provided by parallel experiments with two related alkynes: propyne (CH3CCH) and trifluoropropyne (CF3CCH). Finally, we discuss the implication of these findings to the location of the catalytic site in pMMO.

Identification of the Essential Role of Viral Bcl-2 for Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication.

UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) evades host defenses through tight suppression of autophagy by targeting each step of its signal transduction: by viral Bcl-2 (vBcl-2) in vesicle nucleation, by viral FLIP (vFLIP) in vesicle elongation, and by K7 in vesicle maturation. By exploring the roles of KSHV autophagy-modulating genes, we found, surprisingly, that vBcl-2 is essential for KSHV lytic replication, whereas vFLIP and K7 are dispensable. Knocking out vBcl-2 from the KSHV genome resulted in decreased lytic gene expression at the mRNA and protein levels, a lower viral DNA copy number, and, consequently, a dramatic reduction in the amount of progeny infectious viruses, as also described in the accompanying article (A. Gelgor, I. Kalt, S. Bergson, K. F. Brulois, J. U. Jung, and R. Sarid, J Virol 89:5298-5307, 2015). More importantly, the antiapoptotic and antiautophagic functions of vBcl-2 were not required for KSHV lytic replication. Using a comprehensive mutagenesis analysis, we identified that glutamic acid 14 (E14) of vBcl-2 is critical for KSHV lytic replication. Mutating E14 to alanine totally blocked KSHV lytic replication but showed little or no effect on the antiapoptotic and antiautophagic functions of vBcl-2. Our study indicates that vBcl-2 harbors at least three important and genetically separable functions to modulate both cellular signaling and the virus life cycle. IMPORTANCE: The present study shows for the first time that vBcl-2 is essential for KSHV lytic replication. Removal of the vBcl-2 gene results in a lower level of KSHV lytic gene expression, impaired viral DNA replication, and consequently, a dramatic reduction in the level of progeny production. More importantly, the role of vBcl-2 in KSHV lytic replication is genetically separated from its antiapoptotic and antiautophagic functions, suggesting that the KSHV Bcl-2 carries a novel function in viral lytic replication.